- [Instructor] In this video,
we're gonna show you some tips
for ensuring successful isolation
of high molecular weight DNA from blood samples
using the Monarch High Molecular Weight
DNA Extraction Kit for Cells & Blood.
Unlike many other blood protocols,
the Monarch workflow begins with an erythrocyte lysis step
and we'll review the details of this
so that you know what to expect
at each stage of the process.
We'll begin with fresh blood samples.
After adding three volumes
of cold RBC Lysis Buffer to your sample,
mix immediately by inverting several times.
You'll notice that the solution is opaque.
Incubate on ice until the sample starts lysing,
which will be indicated by the solution
starting to become translucent.
Check the sample frequently,
and each time you check,
invert the sample a few times to mix.
As soon as you notice
that the sample starts to become translucent,
incubate it for an additional three to five minutes on ice
to make sure that the erythrocyte lysis is complete.
The sample is now ready for the centrifugation
at 4 degrees Celsius.
Now let's discuss erythrocyte lysis
with frozen blood samples.
Leukocytes from these samples are very fragile
and tend to clump easily.
If they're not handled properly,
yields can be negatively impacted.
Add three volumes of cold RBC Lysis Buffer
to the frozen blood sample and shake it a few times
to make sure that the buffer
can move freely inside the tube.
Place the samples in a rack at room temperature
and shake them occasionally to monitor their thawing,
until the buffer begins to turn red.
This usually takes around 30 to 90 seconds.
Once the buffer starts to turn red,
place the tubes in a vertical rotating mixer
at about 10 rpm to facilitate thawing
and set a timer to make sure you do not exceed five minutes.
As soon as the samples are thawed,
take them out and if necessary,
invert a few more times manually to complete the thawing.
Vortex the samples briefly
to break up any cell aggregates that may remain
and place them on ice.
You are now ready for the following centrifugation step.
It's very important to work without interruption
and keep the samples on ice to minimize the stress
on the leukocytes during the erythrocyte lysis.
Another important point when processing blood samples,
is that it's essential to remove the supernatant properly
after the erythrocyte lysis
in order to get optimal results.
Carefully remove the supernatant
while keeping the pellet facing down
and keeping the pipette tip on the upper side of the tube.
Leave the last 15 to 20 microliters in the tube
to avoid removing any part of the leukocyte pellet,
which can be relatively loose.
This is what the sample should look like
after the supernatant has been removed properly.
If you remove much, yields will be lower
as you will likely remove a part of the leukocyte pellet.
Leaving too much supernatant will lead
to protein contamination
and may interfere with DNA binding to the beads.
If you are working with several samples,
close each of the caps after supernatant removal
to prevent the pellets from drying out.
The third thing to keep in mind,
is that it's essential that the leukocyte pellets
are completely resuspended at each resuspension step
for the optimal DNA yield and solubility.
There are two ways that you can resuspend the pellet,
you can vigorously flick the tube,
or you can drag the tube forcefully along a tube rack.
We find this to be the most effective approach.
After flicking or dragging to resuspend the pellet,
vortex briefly, add the next buffer,
vortex briefly again to mix,
and check to ensure that the pellet
is resuspended completely.
Complete resuspension is particularly important
during nuclei prep and lysis.
This is because the leukocytes have just undergone
the stressful erythrocyte license procedure.
So, their viability is drastically reduced,
which causes them to be stickier
and more difficult to resuspend.
After the Nuclei Prep Solution is added
and the sample is pipetted up and down a few times,
there may still be some aggregates of leukocytes
that are not easily resuspended
by just pipetting up and down.
Pressing the pipette tip to the bottom of the tube,
while pipetting up and down,
applies additional force that will help
to produce a homogenous nuclei solution.
This will lead to optimal DNA solubility
and the best results.
Our technical support scientists
are always available to help.
Contact us at info@neb.com.